The present invention relates to the analysis of blood to identify and measure properties that correlate with cardiovascular disease.
Cardiovascular disease—primarily in the form of heart attack or stroke—is the leading cause of death in the United States and other developed countries. Cardiovascular disease is likewise becoming an increasing cause of death in developing countries as the risk of death from infectious diseases decreases in such countries.
Some of the main risk factors associated with cardiovascular disease are generally well understood. They include an elevated amount of low density lipoprotein (LDL), high blood pressure, cigarette smoking, diabetes mellitus (“diabetes”), family history, and a less physically active, more sedentary lifestyle.
Serum LDL cholesterol levels are positively correlated with cardiovascular disease risk. However, approximately half of patients who suffer from symptomatic coronary artery disease have normal LDL-cholesterol concentrations. Therefore, there appears to be a hidden risk not detected by conventional clinical laboratory measurements of cholesterol.
As currently best understood, cholesterol deposited in arteries represents a main factor in cardiovascular disease. Cholesterol is effectively insoluble in water and blood and thus the body carries cholesterol using particles called lipoproteins. The body uses several lipid transporting particles present in blood and these lipoprotein particles are typically referred to as chylomicrons, very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL) and high density lipoproteins (HDL). Density increases when less cholesterol is present and density decreases when more cholesterol is present. Thus, the layman often refers to LDL cholesterol as “bad” cholesterol and HDL cholesterol as “good” cholesterol.
Low density lipoprotein particles tend to deposit in artery walls to form atherosclerotic plaques in the artery. In turn, the deposition of LDL to form the atherosclerotic plaques is promoted by an increased LDL concentration (or remnants that can form LDL particles) and a decreased LDL particle size.
In order to help predict and potentially moderate or avoid cardiovascular disease in individuals, conventional clinical tests are carried out to measure certain of the known risk factors. Currently, the most common test is the basic lipid panel which measures total cholesterol, HDL cholesterol (“HDL-C”), and triglycerides. The LDL cholesterol (“LDL-C”) is calculated as the difference between total cholesterol and HDL cholesterol.
Currently, approximately 250,000,000 such tests are carried out in the United States every year, and on a worldwide basis 540 million tests are carried out each year. Current costs are between about $26 and $56 per test.
As an additional factor, LDL can be present in different LDL particle sizes. In turn, smaller LDL particle sizes are associated with an increased risk of cardiovascular disease. Because of the size relationship, information about the size of the LDL particles is valuable in combination with information about the concentration of LDL particles.
Currently, the common tests for measuring LDL particle size include vertical autoprofile (VAPO), gradient gel electrophoresis, and NMR lipoprofiles.
VAP is also referred to as a vertical spin density gradient ultracentrifugation and an exemplary version (“The VAP Cholesterol Test”®) is provided by Atherotech, Inc. of Birmingham, Ala. (USA).
Gradient gel electrophoresis distinguishes particle size in a otherwise conventional electrophoresis (i.e. chromatography) process with an exemplary test offered by Berkeley HeartLab Inc. of (South San Francisco California (USA).
In one commercial embodiment, NMR lipoprofile testing is based upon the chemical shift of the resonant frequencies. LabCorp Inc. (North Carolina, USA) is an exemplary provider of such tests, a number of which are based on U.S. Pat. No. 5,343,389 (and others) to James D. Otvos (“the Otvos patents”). The Otvos patents employ frequency-domain FT-NMR to study lipoprotein particle properties, such as particle size and particle number, in order to perform clinical diagnostic testing and disease risk assessment. In order to provide accurate data, however, chemical shift NMR is typically carried out in large (e.g., 400 megahertz or higher) high resolution Fourier-transform NMR instruments. Many such instruments incorporate a superconducting magnet cooled by a surrounding environment of liquid helium which in turn is surrounded by liquid nitrogen. As a result, the device is large and expensive and the testing is carried out in a small number of central laboratories at a cost of between about 100 and $200 per test.
Such tests also require a frequency-domain analysis, typically performed by a Fourier transform of the data. The key measurable is the chemical shift, a measure of relative frequency and atomic environment. Differences in chemical shifts are used to distinguish and resolve different lipoprotein classes and permit the detection of particle size and number.
In evaluating an individual's lipid profile, core mobility or fluidity of the lipids is a reflection of the relative ratio of different cholesteryl ester and triglyceride molecules in the particle core, which in turn, is a reflection of normal or abnormal lipid metabolism.
Lipoproteins are the body's nanoparticle delivery systems that carry water-insoluble cholesterol and triglyceride molecules through the blood and target them to particular tissues for metabolism. Lipoprotein particles can be distinguished by their density, size, chemical composition and charge. They can also be distinguished by the relative lipid content of the particle's oily core compartment. For example, the cores of LDL and HDL are relatively rich in cholesteryl ester (a highly water-insoluble form of cholesterol), whereas VLDL and chylomicrons are relatively rich in triglycerides. Triglyceride molecules are more flexible than cholesteryl esters, so oil phases rich in triglycerides will appear more fluid and mobile, less viscous. Also, the ratio of these components and thus, the core mobility, changes with metabolism and disease.
On a broad basis, the use of NMR techniques for medical purposes is not new, and the term “NMR” typically can refer to a variety of diagnostic methods. There are many types of NMR methods and instruments and thousands of distinct NMR experiments. A vivid example of this is magnetic resonance imaging (“MRI”), which was originally called NMR Imaging. MRI is a variation of NMR that yields anatomical images rather than chemical signatures. Although MRI is based on the same fundamental physics, it involves different instrumentation, methods and derived measurable from other NMR techniques. Thus, different kinds of NMR are used in somewhat related but distinct areas of medical diagnosis, imaging, and treatment.
U.S. Pat. No. 7,550,971 B2) to Carpenter and Benson describe a method of determining analyte concentrations in body fluids such as blood plasma or serum. Examples given in the claims are the concentrations of glucose, cholesterol, triglycerides, albumin, blood urea nitrogen, alkaline phosphatase and creatinine. The method is restricted to the use of low-field, bench-top TD-NMR instruments, but the measurements and derived quantities are analyte concentration rather than lipoprotein core mobility. These contrasting measurables provide completely different types of diagnostic information.
Arguably, the Carpenter and Benson methods are thinly justified and lack any preliminary data that demonstrates the feasibility of their method for measuring analyte concentrations, and bench-top TD-NMR may not be as suitable for measuring the analyte concentrations as Carpenter and Benson imply. Serum is a complex mixture, and U.S. Pat. No. 7,550,971 lacks any explanation as to how the different analytes in serum can be resolved from one another. Instead, much of the content in U.S. Pat. No. 7,550,971 reflects the known operation of the TD-NMR instrument rather than a technique for resolving analytes from TD-NMR data.
As a result of these various factors, vertical autoprofile, gradient gel electrophoresis, and NMR lipoprofiles can be impractical for routine clinical use; i.e., they are too expensive and too cumbersome to be carried out on-site in a practitioner's office or a hospital laboratory.
As another factor, various lipid tests (e.g., for HDL-C and LDL-C) can be inaccurate in certain circumstances. For example, calculated LDL-C values from a conventional lipid panel are not accurate when determined from non-fasting blood samples or in patients who have elevated triglyceride levels, as is common in diabetes. Likewise, some advanced lipid tests like the NMR LipoProfile require fasting blood samples and thus, cannot monitor changes in lipoprotein particles during metabolism following a meal.
Furthermore, evidence is beginning to emerge that characteristics of lipid-carrying particles other than size and density will correlate with an increased risk of cardiovascular disease.
As yet another factor, when any particular test is difficult to carry out, or must be carried out off-site, or will take significant time to complete, or any combination of these factors, the use of that test will tend to be less frequent than the use of tests that can be carried out quickly and easily at a location—a physician's office, small clinic, or hospital—where patients are typically located and their blood samples taken.
Thus, tests that identify cardiovascular risk and that can be carried out more quickly, more easily, less expensively, and on site would tend to be used more frequently and thus provide greater benefits to individual patients and to the relevant patient population.
Therefore, a need exists for faster, simpler and localized techniques that will identify and measure relevant characteristics that correlate to an expected degree of risk of cardiovascular disease.